Cellular internalization of macromolecules, such as DNA, RNA, proteins, oligonucleotides, and peptides, is still a challenging task because of the presence of the plasma membrane, which constitutes an impermeable barrier for such molecules. Numerous difficulties have been encountered in delivering such molecules to a desired target, including poor penetration into a tissue or cell, toxicity when delivered systemically due to the insufficient specificity of targeting to a particular tissue or cell, side effects when delivered in a high concentration in order to achieve an adequate local concentration at a particular target cell or tissue, and degradation such that inadequate amounts are delivered to the target and/or such that byproducts of degradation result in undesirable side effects.
In order to circumvent these problems, several carrier-mediated delivery systems have been developed. Among them, much attention has recently been given to the use of peptide-based delivery systems. The use of peptides with cell permeability has several advantages, which are mainly due to the various modifications that can be made to the peptide sequence. This allows the engineering of carriers that can address different cellular subdomains and/or are able to transport various types of cargo molecules.
Many cell permeable peptides are designed from sequences of membrane-interacting proteins, such as recombinant proteins, signal peptides, transmembrane domains, and antimicrobial peptides. Within these sequences, short sequences called protein transduction domains (PTDs) have been proved to efficiently cross biological membranes without the need of a carrier or a receptor and to deliver peptides or proteins into intracellular compartments. A number of studies have suggested that the use of PTD-based peptides could be of major importance for therapies against viral diseases or cancers. Among the PTD-based peptides, the third helix of the homeodomain of antennapedia called penetratin (Joliot, A. and A. Prochiantz, Nat. Cell Biol. 6(3):189-96 (2004)), the Tat peptide derived from the transactivating protein Tat of HIV-1 (Wadia, J. S. and S. F. Dowdy, Curr. Opin. Biotechnol. 13(1):52-6 (2002)), transportan (Pooga et al., Faseb J. 12(1):67-77 (1998)), and VP22 (Elliott, G and P. O'Hare, Cell 88(2):223-33 (1997)) have been shown to improve the cellular uptake of peptides, proteins, and oligonucleotides.
A second category of cell-penetrating peptides, called amphipathic peptides, has also been described. An amphipathic molecule can be defined, in short, as consisting of two domains: a hydrophilic (polar) domain and a hydrophobic (nonpolar) domain. For peptides, the amphipathic character can arise from either the primary structure or the secondary structure. Primary amphipathic peptides can be defined as the sequential assembly of a domain of hydrophobic residues with a domain of hydrophilic residues. Secondary amphipathic peptides are generated by the conformational state which allows the positioning of the hydrophobic and hydrophilic residues on opposite sides of the molecule.
Other peptides, such as polyarginine-based peptides, calcitonin-derived peptides, and oligomers, have also been proposed as tools for intracellular delivery of therapeutics.
However, the currently known delivery systems appear to be limited due to their lack of efficiency and/or their toxicity, and little is known about the pathway of their cellular uptake, constituting a handicap for improving their efficiency. In addition, a number of delivery systems are limited in their ability to cross cellular and nuclear membranes. Even where such delivery peptides do cross cell membranes, they are often limited in their efficacies due to their entrapment in endosomes.
The present invention is directed to overcoming these deficiencies in the art.